Prognostic value of CD155/TIGIT expression in patients with colorectal cancer

Introduction The interaction of CD155 with its ligand, the T cell immunoreceptor with Ig and ITIM domains (TIGIT), is being studied owing to its potential to act as a target in the treatment of various solid tumors. However, the relationship between CD155 and TIGIT in colorectal cancer (CRC) prognosis is not known. We hypothesized that the TIGIT–CD155 pathway suppresses the attack of T cells on tumors, thereby affecting CRC prognosis. Methods We examined the expression of CD155 and TIGIT using immunohistochemical staining in 100 consecutive patients with CRC who underwent complete resection of ≤Stage III tumors at Wakayama Medical University Hospital between January and December 2013. We assessed the correlation between CD155 and TIGIT expressions and prognosis as well as the clinicopathological background of CD155 and TIGIT. Results Patients with high CD155 and TIGIT expressions showed worse prognosis than those with other levels of expression (p = 0.026). In multivariate analysis that also included the existing prognostic markers, high CD155 and TIGIT expressions were identified as independent poor prognostic factors. Conclusions The interaction between CD155 and TIGIT possibly plays an important role in the immunological mechanism of CRC. Therefore, it may be possible to effectively predict the postoperative prognosis of CRC by evaluating the combined expression of CD155 and TIGIT. The study findings suggest that CD155 and TIGIT can predict clinical outcomes, thereby contributing to the personalized care of CRC.


Introduction
Colorectal cancer (CRC), particularly colorectal adenocarcinoma, is an important neoplasm with high incidence and mortality [1]. The current treatment approach for CRC is tumorectomy at the early stage and chemotherapy and/or radiation therapy at the advanced stage [2]. In recent years, certain immune checkpoint molecules that belong to the CD28 superfamily, such as programmed cell death 1 (PD 1) and PD ligand 1 (PD-L1), have been reported as potential targets in CRC immunotherapy [3].
In the present study, we focused on the transmembrane protein CD155, which is a nectinlike molecule overexpressed in several cancers [4][5][6]. In malignant melanoma, CD155 deficiency upregulates the expression of DNAX accessory molecule 1 in CD8+ T cells and natural killer (NK) cells, which inhibit tumor growth and metastasis. Furthermore, in recent studies on hepatocellular carcinoma, CD155 was shown to be closely associated with disease exacerbation, indicating its potential for use as a prognostic marker [7,8]. It has also been reported that CD155 functions as a prognostic factor of recurrence and overall survival in small cell lung cancer, pancreatic cancer, bile duct cancer, soft tissue sarcoma, and melanoma [9][10][11][12][13]. Moreover, several studies investigating tumor-expressing proteins have examined the role of the extrinsic regulators of cancer cells in the tumor microenvironment (TME) [14,15]. For instance, the role of T cell immunoreceptor with Ig and ITIM domains (TIGIT) in T cell depletion during binding with CD155 is a focus of immune checkpoint research [16]. In particular, TIGIT overexpression is observed in several malignant tumors, including gastric cancer, noninvasive ductal carcinoma, and melanoma [17][18][19]. However, limited studies have been conducted on the interaction between CD155 and TIGIT in the context of CRC.
We hypothesized that the TIGIT-CD155 pathway suppresses T cell attack on tumors, thereby affecting prognosis. Thus, this study aimed to clarify the clinical importance of CD155 and TIGIT in CRC.

Patients
The subjects comprised 100 consecutive patients who underwent surgical resection for �Stage III CRC at Wakayama Medical University Hospital between January and December 2013. We examined the patients' clinicopathological features, including age, sex, histological grade, TNM staging, follow-up data, neutrophil/lymphocyte ratio (NLR), lymphocyte/monocyte ratio (LMR), modified Glasgow prognostic score (mGPS), CA19-9, and carcinoembryonic antigen (CEA). We did not use surgical specimens and clinicopathological data for research purposes. The study was approved by the Research Ethics Committee of Wakayama Medical University Hospital (approval number: 2747), and patients' verbal consent was obtained. We recorded the patients' clinicopathological features in the patient chart. Clinical specimens were stored and processed in the Department of Human Pathology, whereas clinical pathological data were stored in the Second Department of Surgery, Wakayama Medical University. All cases were followed up for >5 years and a maximum of 7 years.
BOND-III Automated Immunostainer (Leica Microsystems) was used for staining. For the antigen activation method, CD155 was thermally treated (at 100˚C) for 15 min with BOND Epitope Retrieval Solution 1 (Leica Microsystems), whereas TIGIT was thermally treated for 30 min with BOND Epitope Retrieval Solution 2 (Leica Microsystems). The treatment time for the primary antibody was 30 min. The BOND Polymer Refine Detection Kit (Leica Microsystems) was used for DAB staining. Fig 1 shows the outline of the immunohistochemical staining procedure.

Statistical analyses
All statistical analyses were performed using BellCurve for Excel (Social Survey Research Information Co., Ltd). The relationship between CD155 expression in tumor cells, TIGIT expression in tumor-infiltrating immune cells, and clinicopathological features were analyzed via chi-square test. The survival rate was analyzed using the Kaplan-Meier method, and the

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significance of the differences in the survival rate was evaluated using the log-rank test. The variation effect in terms of overall survival was calculated using the Cox regression model. Pvalues < 0.05 were considered statistically significant.

Results
A total of 100 patients with CRC who underwent radical resection were included in this study. Tables 1-3 summarize patient background. A total of 53 (53%) patients were aged > 70 years, and 47 (47%) patients were aged < 70 years. The study group comprised 55 males (55%) and 45 females (45%). With regard to T stage, 15 patients belonged to Tis/1a/1b and 85 belonged to T2/3/4. With regard to N stage, 61 patients belonged to N0 and 39 belonged to N1/2/3. The tumor site was the right colon in 26 cases and the left colon in 74 cases. The histological grade was well-differentiated adenocarcinoma (tub1) in 40 patients and other histological grades in the remaining 60 patients.

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Prognostic value of CD155/TIGIT expression in colorectal cancer patients had an LMR of �2.82 and 89 had an LMR of >2.82. A total of 79 patients had an mGPS of 0 and 21 had an mGPS of 1 or 2. Further, 63 patients had a preoperative CEA value of �5 ng/mL and 37 had a preoperative CEA value of >5 ng/mL. Finally, 93 patients had a preoperative CA19-9 value of �37 U/mL and 7 had a preoperative CA19-9 value of >37 U/mL. CD155 was expressed in malignant CRC epithelial cells but not in normal glandular epithelium (Fig 2A). We also found that TIGIT was highly expressed in the lymphocytes of CRC tissues than in the lymphocytes of adjacent tissues (Fig 2B).

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stage, location, differentiation, NLR, LMR, mGPS, CEA value, and CA19-9 value) were not associated with the expression of CD155 and TIGIT. The Kaplan-Meier survival curve revealed that patients with high CD155 expression had short overall survival (OS) (Fig 5). No significant difference was observed in the survival curves of the high and low TIGIT expression groups (Fig 6). In addition, when the prognostic value of the combined expression of CD155 and TIGIT in determining OS was analyzed, the OS of patients with high CD155 and TIGIT expression was found to be the lowest (Fig 7). The 5-year survival rate for patients with high CD155 expression was 70.7%, and the 5-year survival rate for those with low CD155 expression was 90.6%. The 5-year survival rate for patients with high TIGIT expression was 73.6%, and the 5-year survival rate for those with low CD155 expression was 90.5%. In addition, the 5-year survival rate for patients with high expression of both CD155 and TIGIT was 70.4%, and the 5-year survival rate for other patients was 88.9%.
Univariate Cox proportional hazards regression analysis for OS (Table 4) showed that patients with high CD155 and TIGIT expressions had a higher risk of death than other patients [hazard ratio (HR), 3.272; 95% confidence interval, 1.117-9.589; p = 0.031]. As per PLOS ONE multivariate analysis (Table 5), in addition to lymph node metastasis and elevated CEA, high expressions of CD155 and TIGIT were independent prognostic predictors in patients with CRC. Taken together, these results suggest that a combined analysis of CD155 and TIGIT expressions represents an effective prognostic predictor of CRC.

Discussion
Tumor cells are involved with inhibitory T cell receptors; by weakening T cell function in TME and expressing several ligands, tumors escape from immune surveillance. Therefore, increasing the amount of TILs is important for improving anticancer treatment. Therefore, the use of immune checkpoint inhibitors, including cytotoxic T lymphocyte-associated antigen 4 and PD-1, has been evaluated in clinical trials and shown to prolong patient survival [21][22][23][24]. However, so far, their clinical tumor suppression effects appear to be limited. Therefore, we believe that it is necessary to search for new therapeutic targets. In addition, the role of biochemical and molecular markers in the diagnosis, prognosis, and treatment of malignant tumors in patients with various carcinomas is currently being emphasized [25,26]. Hence, we

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focused on the recently reported CD155-TIGIT pathway. It has been reported that the combined expression level of CD155 and TIGIT correlates with the prognosis of lung adenocarcinoma, gastric cancer, hepatocellular carcinoma, and head and neck squamous cell carcinoma [27][28][29][30].
Few studies have examined the CRC-related prognosis using immunohistochemical staining, and it was hypothesized that similar correlation exists for CRC. In the present study, we evaluated the clinical importance of a recently discovered immunosuppressive ligand, CD155, as well as TIGIT for treating CRC.
Studies have shown that CD155 plays an extremely important role in the development of various malignant tumors, including melanoma, pancreatic cancer, human bile duct cancer, glioblastoma, breast cancer, and primary small cell carcinoma of the esophagus [7,11,[31][32][33]. In the present study, CD155 overexpression was observed in 68% of tissues samples collected

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from patients with CRC, and it was clearly higher in the tumor regions than in the normal regions. Furthermore, CD155-high patients had notably worse prognosis than CD155-low patients. Studies have shown that the expression of CD155 contributes to the postoperative prognosis of patients with malignant tumors, including lung adenocarcinoma and soft tissue sarcoma [12,29,34]. Our data further highlight the importance of CD155 as a therapeutic target in CRC. However, the immunological role of CD155 is complex, and many immune cells other than CRC cells could be affected by treatments that target CD155. Thus, given that there is a chance that acquired immune deficiency, autoimmune disorders, or unknown adverse events could be induced by CD155-targeted therapy, further study is necessary.
TIGIT prevents autoimmune reaction and functions as a checkpoint molecule that helps balance the immune system [35]. In this study, TGIT expression was clearly higher in the

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tumor regions than in the normal regions. Although the expression of TIGIT alone was not associated with prognosis, prognosis was poor in patients with high expression levels of both CD155 and TIGIT.
In multivariate analysis, high expression of CD155 and TIGIT was also proved to be a prognostic marker along with other known prognostic markers. This finding suggests that poor postoperative prognosis can be determined by analyzing the combined expression of CD155 and TIGIT.
To date, limited studies have evaluated the expression of CD155 and TIGIT using immunohistochemical staining in patients with CRC. In the present study, we confirmed a significant difference in the survival curves based on the intensity of CD155 expression but not in those based on the expression of TIGIT alone. These differences are likely caused by other factors in TME, which could not be elucidated by immunohistochemical staining alone. In addition, Qu et al. showed that the high expression of CD155 is associated with good prognosis, which is contrary to our findings [36]. This suggests that it is necessary to standardize CD155 and TIGIT scoring patterns and detection methods to achieve a more reliable and reproducible prediction using the prognostic markers of CRC and other cancers. Furthermore in the multivariate analysis, significant differences were found in the N stages and CEA levels in addition to the high expression of both CD155 and TIGIT. For patients in the advanced stages of CRC, more research is needed to eliminate the effects of bias due to patient treatments.
In conclusion, by analyzing the combined expression of CD155 and TIGIT, it may be possible to effectively predict the postoperative prognosis of patients with CRC. Thus, this combined expression may have potential as a target for CRC therapy. Anti-TIGIT drugs are

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currently being assessed in clinical trials. Furthermore, the group with high CD155 and TIGIT expressions showed poor prognosis. Thus, CD155 and TIGIT expressions may be useful prognostic markers. The study findings suggest that CD155 and TIGIT can predict clinical outcomes, thereby contributing to the development of personalized care for patients with CRC.